The overall goals of this research are: (1) to develop a novel high performance liquid chromatography electrospray/ matrix-assisted laser desorption/ionization-mass spectrometry (HPLC-ESI/MALDI-MS) interface to enhance the mass spectrometric identification and sequence analysis of proteins in complex mixtures and (2) to employ this technology to solve problems at the forefront of biological research. The specific aims are to: (1) develop a novel ionization method for mass spectrometry that is capable of increasing proteome coverage, while at the same time providing a more rapid and inexpensive approach for the analysis of proteins in complex mixtures and (2) to apply developed technology to identify proteins that function as diagnostic markers or potential drug targets for acute myeloid leukemias. By operating at atmospheric pressure, the technique will eliminate many of the barriers encountered when combining MALDI with microcapillary HPLC. The method will utilize ESI aerosol droplets to serve as a platform for MALDI ion generation. And unlike any other HPLC-MS approach, this method will capitalize the favorable attributes of both ionization methods simultaneously. This technology will be applied to determine the regulatory pathways involved with the mixed lineage leukemia gene (MLL), which is a common target for chromosome translocation that often results in acute myeloid leukemias. Utilizing HPLC-ESI/MALDI-MS, we propose to identify proteins, induced by the MLL-ENL fusion protein that could function as biomarkers or potential drug targets for treatment of acute myeloid leukemias.